Use of cytokines and mitogens to inhibit graft versus host disease

ABSTRACT

The field of the invention is generally related to pharmaceutical agents useful in treating graft-versus-host disease (GVHD) in patients that have received allogenic bone marrow transplants.

This application claims the benefit of U.S. Ser. No. 60/076,677, filedMar. 3, 1998.

FIELD OF THE INVENTION

The field of the invention is generally related to pharmaceutical agentsuseful in treating graft-versus-host disease (GVHD) in patients thathave received allogenic bone marrow transplants.

BACKGROUND OF THE INVENTION

Organ transplantation is now used with great success to improve thequality of human life. Substantial progress has been made in usingkidneys, hearts, and livers from unrelated individuals. However,transplantation of hematopoietic stem cells from an unrelated (orallogeneic) donor is a more complicated endeavor. Here multipotent stemcells which have the capacity to regenerate all the blood-formingelements and the immune system are harvested from bone marrow orperipheral blood from one individual are transferred to another.However, histocompatibility differences between donor and recipientresults in a higher incidence of transplant-related complications, andhas limited the use of this procedure (Forman et al., BlackwellScientific Publications, 1994).

It is unfortunate that only few individuals are candidates forallogeneic hematopoietic stem cell transplantation at the present timebecause the spectrum of diseases treatable by this procedure hassteadily increased. These diseases now include hematologic malignanciessuch as the acute or chronic leukemias, multiple myeloma,myelodysplastic syndromes; lymphomas; and the severe anemias such asaplastic anemia or thalassemia.

Allogeneic stem cell transplantation begins with treatment of therecipient with a highly immunosuppressive conditioning regimen. This ismost commonly accomplished with high doses of chemotherapy and radiationwhich effectively kill all the blood forming elements of the bonemarrow. Besides preparing the recipient bone marrow for donor stem celltransplantation, the conditioning regimen serves to kill much of themalignancy that remains in the body. The period of time between thecompletion of the conditioning regimen, and engraftment of the donorstem cells is the most dangerous for the recipient. It is during thistime that the patient is completely immunocompromised and susceptible toa host of life-threatening infections. This vulnerability persists untilthe grafted donor stem cells proliferate and differentiate into theneeded white blood cells and immune cells needed to combat infections.

Moreover, donor stem cell preparations generally contain immune cellscalled T lymphocytes. Unless the donor stem cells originate from anidentical twin the transferred T cells turn against the recipient'stissues and trigger a deadly ailment called graft versus host disease(or GVHD). This is because the donor T lymphocytes recognizehistocompatibility antigens of the recipient as foreign and respond bycausing multi-organ dysfunction and destruction.

Current techniques of immunosuppression have made allogeneic stem celltransplantation from a related, histocompatible (HLA-matched) donor muchsafer than it once was. Allogeneic stem cell transplantation from anunrelated, HLA-matched donor is commonly complicated by serious, oftenfatal GVHD. The threat of GVHD is even higher when the stem cell donoris HLA mismatched.

Since only 30% of patients in need of allogeneic stem cells will have asibling with identical histocompatibility antigens (Dupont, B., ImmunolReviews 157:12, 1997), there is a great need to make HLA-matchedunrelated, and HLA-mismatched transplantation a safer procedure. Therehave been two principal approaches to resolving this problem. The firsthas been to deplete the graft of contaminating T lymphocytes and thesecond has been to inactivate the T cells so they cannot attack therecipient.

In the 1970's it became evident that ex-vivo removal of mature Tlymphocytes from a bone marrow graft prior to transplantationdramatically decreased or prevented GVHD in animals receiving marrowgrafts across major histocompatibility barriers (Rodt, H. J. Immunol4:25-29, 1974; and 4 Vallera et al., Transplantation 31:218-222, 1981).However, with T cell depletion the incidence of graft failure, graftrejection, relapse of leukemia, and viral-induced lympho-proliferativedisease markedly increased (Martin et al. Blood 66:664-672, 1985; 6Patterson et al. Br J Hematol 63:221-230, 1986; Goldman et al. Ann IntemMed 108:806-814, 1988; and Lucas et al. Blood 87:2594-2603, 1996). Thus,the transplantation of donor T cells on the stem cells has beneficial aswell as deleterious effects. One needs the facilitating effect of the Tcells on the engraftement of stem cells and the now well recognizedgraft-versus-tumor effects, but not graft-versus host disease.

Several approaches have been used to decrease T cell activation. Theseinclude: 1) in vivo immunosuppressive effects of drugs such as FK506 andrapamycin (Blazar et al. J. Immunol 153:1836-1846, 1994; Dupont et al.J. Immunol 144:251-258, 1990; Morris, Ann NY Acad Sci 685:68-72. 1993;and Blazar et al. J Immunol 151:5726-5741, 1993); 2) the in vivotargeting of GVHD-reactive T cells using intact and F(ab′)2 fragments ofmonoclonal antibodies(mAb)reactive against T cell determinants or mAblinked to toxins (Gratama et al. Amj Kidney Dis 11:149-152, 1984; Hirumaet al. Blood 79:3050-3058, 1992; Anasetti et al. Transplantation54:844-851, 1992; Martin et al. Bone Marrow Transplant 3:437-444, 1989);3) inhibition of T cell signaling via either IL-2/cytokine receptorinteractions (Herve et al. Blood 76:2639-2640, 1990) or the inhibitionof T cell activation through blockade of co-stimulatory or adhesogenicsignals (Boussiotis et al. J Exp Med 178:1753-1763, 1993; Gribben et al.Blood 97:4887-4893, 1996; and Blazar et al. Immunol Rev 157:79-90,1997); 4) the shifting of the balance between acute GVHD-inducing Thelper-type 1 T cells to anti-inflammatory T helper-type 2 T cells viathe cytokine milieu in which these cells are generated (Krenger et al.Transplantation 58:1251-1257, 1994; Blazar et al. Blood 88:247, 1996,abstract; Krenger et al. J Immunol 153:585-593. 1995; Fowler et al.Blood 84:3540-3549, 1994); 5) the regulation of alloreactive T cellactivation by treatment with peptide analogs which affect either T cellreceptor/major histocompatibility complex (MHC) interactions, class IIMHC/CD4 interactions, or class I MHC/CD8 interactions (Townsend andKorngold (unpublished data)); and 6) the use of gene therapy to halt theattack of donated cells on the recipient's tissues (Bonini et al.Science 276:1719-24, 1997).

There is suggestive evidence that the T lymphocytes from non-identicaldonors can become tolerant to the recipient's tissues. Unlike patientswho receive solid organ allografts for whom life-long immunosuppressivetherapy is needed to control chronic rejection, there is evidence ofimmunologic tolerance with stem cell allografts. The majority of thesepatients can be withdrawn from immune suppression without furtherevidence of GVHD (Storb et al. Blood 80:560-561, 1992; and Sullivan etal. Semin Hematol 28:250-259, 1992).

Immunologic tolerance is a specific state of non-responsiveness to anantigen. Immunologic tolerance generally involves more than the absenceof an immune response; this state is an adaptive response of the immunesystem, one meeting the criteria of antigen specificity and memory thatare the hallmarks of any immune response. Tolerance develops more easilyin fetal and neonatal animals than in adults, suggesting that immature Tand B cells are more susceptible to the induction of tolerance.Moreover, studies have suggested that T cells and B cells differ intheir susceptibility to tolerance induction. Induction of tolerance,generally, can be by clonal deletion or clonal anergy. In clonaldeletion, immature lymphocytes are eliminated during maturation. Inclonal anergy, mature lymphocytes present in the peripheral lymphoidorgans become functionally inactivated.

Following antigenic challenge stimulation, T cells generally arestimulated to either promote antibody production or cell-mediatedimmunity. However, they can also be stimulated to inhibit these immuneresponses instead. T cells with these down-regulatory properties arecalled “suppressor cells”.

Although it is known that T suppressor cells produce cytokines such astransforming growth factor beta (TGF-beta), interleukin 4 (IL-4) orinterleukin (IL-10) with immunosuppressive effects, until recently themechanisms responsible for the generation of these regulatory cells havebeen poorly understood. It was generally believed that CD4+ T cellsinduce CD8− T cells to develop down-regulatory activity and thatinterleukin 2 (IL-2) produced by CD4+ cells mediates this effect.Although most immunologists agree that IL-2 has an important role in thedevelopment of T suppressor cells, whether this cytokine works directlyor indirectly is controversial (Via et al. International Immunol5:565-572, 1993; Fast, J Immunol 149:1510-1515, 1992; Hirohata et al. JImmunol 142:3104-3112, 1989; Taylor, Advances Exp Med Biol 319:125-135,1992; and Kinter et al., Proc. Nati. Acad. Sci. USA92:10985-10989,1995). Recently, IL-2 has been shown to induce CD8+ cellsto suppress HIV replication in CD4−T cells by a non-lytic mechanism.This effect is cytokine mediated, but the specific cytokine with thiseffect has not been identified (Barker et al. J Immunol 156:4476-83,1996 ; and Kinter et al. Proc Nat Acad Sci USA 99:10985-9 1995).

A model using human peripheral blood lymphocytes to study T cell/B cellinteractions in the absence of other accessory cells has been developed(Hirokawa et al. J. Immunol. 149:1859-1866, 19??). With this model itwas found that CD4+ T cells by themselves generally lacked the capacityto induce CD8+ T cells to become potent suppressor cells. Thecombination of CD8+ T cells and NK cells, however, induced strongsuppressive activity (Gray et al. J Exp Med 180:1937-1942, 1994). It wasthen demonstrated that the contribution of NK cells was to produceTGF-beta in its active form. It was then reported that a smallnon-immunosuppressive concentration (10-100 pg/ml) of this cytokineserved as a co-factor for the generation of strong suppressive effectson IgG and IgM production (Gray et al. J Exp Med 180:1937-1942, 1994).Further, it was demonstrated that NK cells are the principal lymphocytesource of TGF-beta (Gray et al. J Immunol, 160:2248-2254, 1998).

TGF-beta is a multifunctional family of cytokines important in tissuerepair, inflammation and immunoregulation (Border et al. J Clin Invest90:1-7, 1992; and Sporn et al. J Cell Biol 105:1039-1045, 1987).TGF-beta is unlike most other cytokines in that the protein released isbiologically inactive and unable to bind to specific receptors(Massague, Cell 69:1067-1070, 1992). The conversion of latent to activeTGF-beta is the critical step which determines the biological effects ofthis cytokine.

There is some evidence that NK cell-derived TGF-beta has a role in theprevention of GVHD. Whereas the transfer of stem cells from one strainof mice to another histocompatibility mismatched strain resulted indeath of all recipients from GVHD within 19 days, the simultaneoustransfer of NK cells from the donor animals completely prevented thisconsequence. All the recipient mice survived indefinitely. Thistherapeutic effect, however, was completely blocked by antagonizing theeffects of TGF-beta by the administration of a neutralizing antibody(Murphy et al. Immunol Rev 157:167-176, 1997).

It is very likely, therefore, that the mechanism whereby NK cell-derivedTGF-beta prevented GVHD is similar to that described by Horwitz et al.in the down-regulation of antibody production. In each case NKcell-derived TGF-beta was responsible for the generation of suppressorlymphocytes that blocked these respective immune responses. The mousestudy is of particular interest since the histocompatibility differencesbetween genetically disparate inbred mice strains would mirror that ofunrelated human donors. A modification of this strategy, therefore mightovercome GVHD in mismatched humans.

Anti-CD2 monoclonal antibodies and other constructs that bind to the CD2co-receptor have been shown to be immunosupressive. It has now beendemonstrated that at least one mechanism to explain thisimmunosuppressive effect is by inducing the production of TGF-beta (Grayet al. J Immunol, 160:2248-2254, 1998).

One strategy to prevent GVHD would be to isolate and transfer NK cellsalong with the stem cells. Another would be to treat theimmunocompromised recipient who has received allogeneic stem cells withTGF-beta, anti-CD2 monoclonal antibodies, IL-2 or a combination of thesecytokines. The first strategy would be difficult because NK cellscomprise only 10 to 20% of total lymphocytes so that it would bedifficult to harvest a sufficient number of cells for transfer. Thesecond strategy is limited by numerous effects on different body tissuesand are not very safe to deliver to a patient systemically. What isneeded, therefore, is a way to induce mammalian cells to suppress thedevelopment of GVHD ex vivo.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides methods for inducing T cell tolerance in a sample of ex vivoperipheral blood mononuclear cells (PBMCs) comprising adding asuppressive composition to the cells. The suppressive composition can beIL-10, TGF-β, or a mixture.

In an additional aspect, the present invention provides methods fortreating donor cells to ameliorate graft versus host disease in arecipient patient. The methods comprise removing peripheral bloodmononuclear cells (PBMC) from a donor, and treating the cells with asuppressive composition for a time sufficient to induce T celltolerance. The cells are then introducing to a recipient patient. ThePBMCs can be enriched for CD8+ cells, if desired. The methods mayadditionally comprising adding the treated cells to donor stem cellsprior to introduction into the patient.

In an additional aspect, the invention provides kits for the treatmentof donor cells comprising a cell treatment container adapted to receivecells from a donor and at least one dose of a suppressive composition.The kits may additionally comprising written instructions and reagents.The cell treatment container may comprise a sampling port to enable theremoval of a fraction of the cells for analysis, and an exit portadapted to enable transport at least a portion of the cells to arecipient patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict that TGF-β can upregulate expression of CD40Ligand (CD40L) on T cells. Purified T cells were stimulated with PMA (20ng/ml) and ionomycin (5 μM) in the presence or absence of TGF-β. After 6hours the cells were stained with anti-CD40L antibodies. In the absenceof TGF-β, there were 30% positive cells (solid line, panel A). With 100pg/ml of TGF-β, 66% of the cells were positive (solid line, panel B).The dotted line in both panenis is the reactivity of a control antibody.

FIGS. 2A, 2B, 2C and 2D depict that TGF-β increases TNF-α expression byCD8+ cells. Purified CD8+ cells were stimulated for 24 hours with Con A(5 pg/ml) + TGF-β (10 pg/ml)+ IL-2 (10 U). During the last 6 hours,monensin (2 μM) was also present to prevent cytokine release. The cellswere first stained with anti-CD69 to distinguish the activated cells.Then the cells were fixed (4% paraformaldhyde), permeabilized (0.1%saponin) and stained with anti-TNF-α antibodies.

FIGS. 3A and 3B depict TGF-β, enhances IL-2 expression by T cells.Purified T cells were stimulated in the presence or absence of TGF-β (1ng/ml). In the absence of TGF-β, 36% of the cells were positive (panelA, solid line) whereas with TGF-β, 53% were positive (panel B, solidline).

FIGS. 4A, 4B and 4C depict that TGF-β can enhance or inhibit cytotoxicactivity. In panels A and B, purified T cells were cultured withirradiated allogenic stimulator cells in the presence or absence of theindicated cytokines. After 48 hours, the cells were washed and after afurther 3 days, assayed for cytotoxic activity against ³¹Cr-labelledstimulator ConA blasts. In panel C, purified CD8+ cells were culturedwith irradiated allogenic cells in the presence or absence of TGF-β(10pg/ml) or IL-12 (100 U). After 48 hours, the cells were washed and addedto autologous T cells and irradiated allogenic cells. After 5 days ofculture, cytotoxic activity was determined using ³¹Cr-labelledstimulator ConA blasts as target cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention allows for the transfer of histoincompatible stemcells to humans with a variety of malignant or hereditary diseases usinga method to prevent life-threatening graft-versus-host disease. This isaccomplished by treatment of donor cells with a combination of mitogensand cytokines ex-vivo. The particular advantage of this procedure isthat it avoids the removal of donor T cells which facilitate stem cellengraftment and that have the potential to attack any remainingmalignant cells. Once a state of tolerance between donor and host hasbeen achieved, non-conditioned donor T cells can be transferred tomaximize the beneficial graft-versus-tumor immune response.

This strategy is unlike almost all other treatment modalities currentlyin use. These cytokines and mitogens described would have severe toxicside effects if administered in vivo. The ex-vivo protocol describedavoids these side effects. The ability to successfully engrafthistoincompatible stem cells for treatment of life-threatening diseaseswould be a milestone in medicine.

In addition, a further advantage of the present invention is that it mayavoid or minimize the very toxic immunosuppressive medicines that mustbe given to the recipient to prevent GVHD. These medicines also blockthe ability of the donor-derived lymphocytes which repopulate the immunesystem of the recipient from becoming “educated” to their new host.Therefore, it is difficult to stop the immunosuppressive drugs, unlessan alternative treatment such as the present invention is used.

The strategy of the present invention is to suppress GVHD by bothsuppressing T cell activation and inducing a tolerant state in the donorcells, which prevents the donor cells from attacking recipient cells.Surprisingly, the methods outlined herein result in not only thesuppression of the treated cells but additionally induces them toprevent other donor cells from killing recipient cells as well, i.e.they become tolerant. That is, the methods outlined herein not onlydecrease the capacity of the donor's cells to attack the recipient'scells, but induces some of the donor's cells to assume a surveillancerole and prevent other donor cells from mounting an immune attackagainst the recipient host. The net result is for the donor lymphocytesto be tolerant to the histocompatibility antigens of the recipient, butdoes not impair the ability of the new lymphocytes to attack tumorcells.

Another significant potential advantage of this strategy is a lowprobability of serious adverse side effects. Since only trace amounts ofsuppressive compositions such as cytokines will be returned to thepatient, there should be minimal toxicity.

Accordingly, the present invention is drawn to methods of treating donorcells for transplantation into a recipient that comprise removingperipheral blood mononuclear cells (PBMCs) from the donor and treatingthe cells with a composition that is on one hand suppressive, but on theother hand generates surveillance cells to prevent an immune attack.

The present invention shows that the treatment of the donor cells by asuppressive composition blocks an immune attack against the recipient'scells. Without being bound by theory, it appears that there are severalways the methods of the invention may work. First of all, the donorcells are activated to become tolerant to the recipient's cells.Secondly, the donor CD8+ cells get activated to become regulatory cells,to prevent other donor cells from killing recipient cells. These resultslead to amelioration of a GVH response. Without being bound by theory,it appears that the inhibition of cytotoxic activity may occur as aresult of the effects TGF-β on the cells; as depicted in the figures,the addition of TGF-β causes the upregulation of CD40L on T cells,increases TNF-α expression by CD8+ cells, and enhances IL-2 expression.

Thus, in a preferred embodiment, the present invention induces tolerancein the donor cells to recipient tissue, thus avoiding GVHD, by treatingthem with a suppressive composition ex vivo.

Accordingly, the present invention provides methods of treating donorcells to induce or establish tolerance to recipient cells prior totransplantation into a recipient patient to decrease or eliminate agraft-versus-host response. By “T cell tolerance” herein is meant immunenon-responsiveness to the recipient, i.e. a tolerance to thehistocompatibility antigens of the recipient. Without being bound bytheory, this may be due to anergy or death of the T cells. Preferably,the T cells retain the ability to recognize other antigens as foreign,to facilitate tumor killing and general immunological responses toforeign antigens.

Using the methods outlined herein, a GVH response is suppressed ortreated. By “treating” GVHD herein is meant that at least one symptom ofthe GVHD is ameliorated by the methods outlined herein. This may beevaluated in a number of ways, including both objective and subjectivefactors on the part of the patient as is known in the art. For example,GVHD generally exhibits a skin rash, an abnormality in liver functionstudies, fever, general symptoms including fatigue, anemia, etc.

By “patient” herein is meant a mammalian subject to be treated, withhuman patients being preferred. In some cases, the methods of theinvention find use in experimental animals and in the development ofanimal models for disease, including, but not limited to, rodentsincluding mice, rats, and hamsters; and primates.

The methods provide for the removal of blood cells from a patient. Ingeneral, peripheral blood mononuclear cells (PBMCs) are taken from apatient using standard techniques. By “peripheral blood mononuclearcells” or “PBMCs” herein is meant lymphocytes (including T-cells,B-cells, NK cells, etc.) and monocytes. As outlined more fully below, itappears that the main effect of the suppressive composition is to enableCD8+ T cells to become tolerant. Accordingly, the PBMC population shouldcomprise CD8+ T cells. Preferably, only PBMCs are taken, either leavingor returning red blood cells and polymorphonuclear leudocytes to thepatient. This is done as is known in the art, for example usingleukophoresis techniques. In general, a 5 to 7 liter leukophoresis stepis done, which essentially removes PBMCs from a patient, returning theremaining blood components. Collection of the cell sample is preferablydone in the presence of an anticoagulant such as heparin, as is known inthe art.

In general, the sample comprising the PBMCs can be pretreated in a widevariety of ways. Generally, once collected, the cells can beadditionally concentrated, if this was not done simultaneously withcollection or to further purify and/or concentrate the cells. The cellsmay be washed, counted, and resuspended in buffer.

The PBMCs are generally concentrated for treatment, using standardtechniques in the art. In a preferred embodiment, the leukophoresiscollection step results a concentrated sample of PBMCs, in a sterileleukopak, that may contain reagents or doses of the suppressivecomposition, as is more fully outlined below. Generally, an additionalconcentration/purification step is done, such as FicollHypaque densitygradient centrifugation as is known in the art.

In a preferred embodiment, the PBMCs are then washed to remove serumproteins and soluble blood components, such as autoantibodies,inhibitors, etc., using techniques well known in the art. Generally,this involves addition of physiological media or buffer, followed bycentrifugation. This may be repeated as necessary. They can beresuspended in physiological media, preferably AIM-V serum free medium(Life Technologies) (since serum contains significant amounts ofinhibitors of TGF-β) although buffers such as Hanks balanced saltsolution (HBBS) or physiological buffered saline (PBS) can also be used.

Generally, the cells are then counted; in general from 1×10⁹ to 2×10⁹white blood cells are collected from a 5-7 liter leukophoresis step.These cells are brought up roughly 200 mls of buffer or media.

In a preferred embodiment, the PBMCs may be enriched for one or morecell types. For example, the PBMCs may be enriched for CD8+ T cells,CD4+ T cells or, in the case of stem cell isolation as is more fullydescribed below, CD34+ stem cells. This is done as is known in the art,as described in Gray et al. (1998), J. Immunol. 160:2248, herebyincorporated by reference. Generally, this is done using commerciallyavailable immunoabsorbent columns, or using research procedures (thePBMCs are added to a nylon wool column and the eluted, nonadherent cellsare treated with antibodies to CD4, CD16, CD11b, and CD74, followed bytreatment with immunomagnetic beads, leaving a population enriched forCD8+ T cells). In one embodiment, cell populations are enriched for CD8+cells, as these appear to be the cells most useful in the methods of theinvention. However, one advantage of using PBMCs is that other celltypes within the PBMC population produce IL-10, thus decreasing or eveneliminating the requirement of the suppressive composition comprisingIL-10.

Once the cells have undergone any necessary pretreatment, the cells aretreated with a suppressive composition. By “treated” in this contextherein is meant that the cells are incubated with the suppressivecomposition for a time period sufficient to result in T cell tolerance,particularly when transplanted into the recipient patient. Theincubation will generally be under physiological temperature.

By “suppressive composition” or “tolerance composition” is meant acomposition that can induce T cell tolerance. Generally, thesecompositions are cytokines. Suitable suppressive compositions include,but are not limited to, IL-10, IL-2 and TGF-β. A preferred suppressivecomposition is a mixture of IL-10 and TGF-β.

The concentration of the suppressive composition will vary on theidentity of the composition, but will generally be at physiologicconcentration, i.e. the concentration required to give the desiredeffect, i.e. an enhancement of specific types of regulatory cells. In apreferred embodiment, TFG-β is used in the suppressive composition. By“transforming growth factor -β ” or “TGF-β” herein is meant any one ofthe family of the TGF-βs, including the three isoforms TGF-β1, TGF-β2,and TGF-β3; see Massague, (1980), J. Ann. Rev. Cell Biol 6:597.Lymphocytes and monocytes produce the β1 isoform of this cytokine (Kehrlet al. (1991), Int J Cell Cloning 9:438-450). The TFG-0 can be any formof TFG-β that is active on the mammalian cells being treated. In humans,recombinant TFG-β is currently preferred. A human TGF-β2 can bepurchased from Genzyme Pharmaceuticals, Farmington, Mass. In general,the concentration of TGF-β used ranges from about 2 picograms/ml of cellsuspension to about 2 nanograms, with from about 10 pg to about 500 pgbeing preferred, and from about 50 pg to about 150 pg being especiallypreferred, and 100 pg being ideal.

In a preferred embodiment, IL-10 is used in the suppressive composition.The IL-10 can be any form of IL-10 that is active on the mammalian cellsbeing treated. In humans, recombinant IL-10 is currently preferred.Recombinant human IL-10 can be purchased. In general, the concentrationof IL-10 used ranges from about 1 U/ml of cell suspension to about 100,with from about 5 to about 50 being preferred, and with 10 U/ml beingespecially preferred.

In a preferred embodiment, IL-2 is used as the suppressive composition.The IL-2 can be any form of IL-2 that is active on the mammalian cellsbeing treated. In humans, recombinant IL-2 is currently preferred.Recombinant human IL-2 can be purchased from Cetus, Emeryville, Calif.In general, the concentration of IL-2 used ranges from about 1 Unit/mlof cell suspension to about 100 U/ml, with from about 5 U/ml to about 25U/ml being preferred, and with 10 U/ml being especially preferred. In apreferred embodiment, IL-2 is not used alone.

In a preferred embodiment, TGF-β can be used alone as the suppressivecomposition. Alternate preferred embodiments utilize IL-10 alone,combinations of TGF-β, IL-10 and IL-2, with the most preferredembodiment utilizing a mixture of TGF-β and IL-10.

The suppressive composition is incubated with the donor cells and apopulation of irradiated PMBC recipient cells (harvested as outlinedabove). The recipient cells are irradiated so that they cannot attackthe donor cells, but will stimulate the donor cells to become tolerantto the recipient cells. The incubation occurs for a period of timesufficient to cause an effect, generally from 4 hours to 96 hours,although both shorter and longer time periods are possible.

In one embodiment, treatment of the donor cells with the suppressivecomposition is followed by immediate transplantation into the recipientpatient, generally after the cells have been washed to remove thesuppressive composition.

In a preferred embodiment, a second step is done. In this embodiment,after the donor cells have been conditioned or treated with thesuppressive composition, they may be frozen or otherwise stored. Then asecond step comprising obtaining a population of donor hematopoieticstem cells from aspirated bone marrow or PBMCs. Stem cells comprise avery small percentage of the white blood cells in blood, and areisolated as is known in the art, for example as described in U.S. Pat.Nos. 5,635,387 and 4,865,204, both of which are incorporated byreference in their entirety, or harvested using commercial systems suchas those sold by Nexell. As outlined above, CD34+ stem cells can beconcentrated using affinity columns; the eluted cells are a mixture ofCD34+ stem cells and lymphocytes. The contaminating lymphocytes aregenerally be removed using known techniques such as staining withmonoclonal antibodies and removal using conventional negative selectionprocedures.

Once the CD34+ stem cells have been isolated, they may be mixed with thedonor cells previously treated with the suppressive composition andimmediated introduced into the recipient patient.

In one embodiment, the cells are treated for a period of time, washed toremove the suppressive composition, and may additionally reincubated.The cells are preferably washed as outlined herein to remove thesuppressive composition. Further incubations for testing or evaluationmay also be done, ranging in time from a few hours to several days. Ifevaluation of any cellular characteristics prior to introduction to apatient is desirable, the cells may be incubated for several days toseveral weeks to expand numbers of suppressor cells.

Once the cells have been treated, they may be evaluated or tested priorto transplantation into the patient. For example, a sample may beremoved to do: sterility testing; gram staining, microbiologicalstudies; LAL studies; mycoplasma studies; flow cytometry to identifycell types; functional studies, etc. Similarly, these and otherlymphocyte studies may be done both before and after treatment. Apreferred analysis is a test using labeled recipient cells; incubatingthe treated tolerant donor cells with a labeled population of therecipient cells will verify that the donor cells are tolerant and won'tkill the recipient cells.

In a preferred embodiment, the treatment results in the conditioning ofthe T cells to become non-responsive to histocompatibility antigens ofthe recipient so that GVHD is prevented.

In a preferred embodiment, prior to transplantation, the amount of totalor active TGF-β can also be tested. As noted herein, TGF-β is made as alatent precursor that is activated post-translationally.

After the cell treatment, the donor cells are transplanted into therecipient patient. The MHC class I and class II profiles of both thedonor and the recipient are determined. Preferably, a non-related donoris found that preferably matches the recipients HLA antigens, but maymismatch at one or more loci if a matched donor cannot be identified.The recipient patient has generally undergone bone marrow ablation, suchas a high dose chemotherapy treatment, with or without total bodyirradiation.

The donor cells are transplanted into the recipient patient. This isgenerally done as is known in the art, and usually comprises injectingor introducing the treated cells into the patient as will be appreciatedby those in the art. This may be done via intravascular administration,including intravenous or intraarterial administration, intraperitonealadministration, etc. For example, the cells may be placed in a 50 molFenwall infusion bag by injection using sterile syringes or othersterile transfer mechanisms. The cells can then be immediately infusedvia IV administration over a period of time, such as 15 minutes, into afree flow IV line into the patient. In some embodiments, additionalreagents such as buffers or salts may be added as well.

After reintroducing the cells into the patient, the effect of thetreatment may be evaluated, if desired, as is generally outlined aboveand known in the art.

The treatment may be repeated as needed or required. After a period oftime, the leukemic cells may reappear. Because the donor lymphocytes arenow tolerant to the recipient's cells, the patient now receives atransfusion of unconditioned donor lymphocytes which recognize theleukemic cells as foreign and kill these cells.

In a preferred embodiment, the invention further provides kits for thepractice of the methods of the invention, i.e., the incubation of thecells with the suppressive compositions. The kit may have a number ofcomponents. The kit comprises a cell treatment container that is adaptedto receive cells from a donor. The container should be sterile. In someembodiments, the cell treatment container is used for collection of thecells, for example it is adaptable to be hooked up to a leukophoresismachine using an inlet port. In other embodiments, a separate cellcollection container may be used.

The form and composition of the cell treatment container may vary, aswill be appreciated by those in the art. Generally, the container may bein a number of different forms, including a flexible bag, similar to anIV bag, or a rigid container similar to a cell culture vessel. It may beconfigured to allow stirring. Generally, the composition of thecontainer will be any suitable, biologically inert material, such asglass or plastic, including polypropylene, polyethylene, etc. The celltreatment container may have one or more inlet or outlet ports, for theintroduction or removal of cells, reagents, suppressive compositions,etc. For example, the container may comprise a sampling port for theremoval of a fraction of the cells for analysis prior to introductioninto the recipient patient. Similarly, the container may comprise anexit port to allow introduction of the cells into the recipient patient;for example, the container may comprise an adapter for attachment to anIV setup.

The kit further comprises at least one dose of a suppressivecomposition. “Dose” in this context means an amount of the suppressivecomposition such as cytokines, that is sufficient to cause an effect. Insome cases, multiple doses may be included. In one embodiment, the dosemay be added to the cell treatment container using a port;alternatively, in a preferred embodiment, the dose is already present inthe cell treatment container. In a preferred embodiment, the dose is ina lyophilized form for stability, that can be reconstituted using thecell media, or other reagents.

In some embodiments, the kit may additionally comprise at least onereagent, including buffers, salts, media, proteins, drugs, etc. Forexample, mitogens can be included.

In some embodiments, the kit may additional comprise writteninstructions for using the kits.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

EXAMPLES Example 1 Donor Lymphocyte ex vivo Treatment to Prevent anImmune Attack Against Blood Cells from an Unrelated Recipient

A blood sample from a donor was obtained and lymphocytes prepared bydensity gradient centrifugation. T cells were prepared using aconventional negative selection procedure. These T cells wereconditioned to prevent them from attacking the recipient cells. For thisconditioning, the CD8+ T cells were mixed with irradiated stimulatorcells from the recipient. The stimulator cells were derived from Tcell-depleted blood cells from the recipient. The mixture of donor Tcells and recipient stimulator cells were cultured for 48 hours withdifferent concentrations of one or more cytokines. In this example thecytokines were TGF-β and IL-10. This procedure abolished the potentialof the donor T cells to kill recipient cells, in FIG. 4B.

To test the ability of the donor T cells to recognize and kill recipientblood cells, the donor T cells were cultured with irradiated stimulatorcells for 5 days. Then the donor cells were cultured for 4 hours with asample of recipient radiolabeled blood cells. When the recipient's cellsare killed they release radioisotope into the culture medium. Bydetermining the amount of radioisotope released, one can calculate thepercentage of cells killed. In the standard cytotoxicity assay shown inFIG. 1, donor cells were cultured with labeled recipient cells in 30 to1, 15 to 1, and 7.5 to 1 ratios. These combinations of donor andrecipient cells are called effector to target ratios. Killing isindicated by the various symbols. As expected, maximum killing was seenat the highest effector to target cell ratio. In panel A, the opencircles shows that if 30 donor cells were mixed with 1 recipient cell,40 percent of the recipient cells were killed. When donor T cells wereconditioned with very small concentrations of TGF-β(0.01 or 0.1nanograms per ml), they had no effect on killing. However, if the Tcells were treated with 1 nanogram per ml of TGF-β, the killing ofrecipient cells decreased by 50 percent. Panel B shows that if the Tcells were treated with IL-10, killing also decreased by 50%. If the Tcells had been conditioned with both IL-10 and TGF-β at 1 nanogram perml, these cells completed blocked the killing of recipient cells;killing was almost undetectable. Various combinations of mitogens,cytokines, and monoclonal antibodies can be used to make T cellsnon-responsive.

Example 2 CD8+ T Cells from the Donor Conditioned ex vivo to Preventother Donors T Cells from Mounting an Attack Against Blood Cells from anUnrelated Recipient

A blood sample from a donor was obtained and lymphocites prepared. CD8+T cells were mixed with irradiated stimulator cells of the recipient andeither TGF-β(picograms per ml) or IL-12 100 U/ml. IL-12 is known toenhance the ability of CD8+ T cells to develop the capacity to kill.Here IL-12 was used to show that a given population of CD8+ cells can beinduced to kill or to block killing depending upon how they areactivated. Other CD8 + cells were cultured in culture medium only as acontrol (CD8med).

The CD8+ T cells, the stimulator cells and the cytokines were culturedfor 48 hours and the cytokines were removed from the cultures bywashing. This procedure not only abolished the potential of the TGF-β,conditioned CD8+ cells to kill the recipient cells, but also inducedthem to prevent other donor T cells from killing the recipient cells(FIG. 4C).

To enable the donor T cells to recognize and kill recipient blood cells,the donor cells were cultured with irradiated stimulator cells for fivedays. Then the donor cells were cultured for 4 hours with a sample ofrecipient radiolabeled blood cells. The open circles show the level ofdonor cells of recipient cells when no CD8+ cells were added. At a 30:1effector to target cell ration, 30% of the recipient's cells werekilled. If CD8+ cells that had been cultured for 48 hours withoutcytokines were added, there was no change in the killing (CD8+ +Med,solid circles). If the CD8+ cells had been conditioned with TGF-β,killing was suppressed by about 50%. However, the CD8+ T cellsconditioned with TGF-β not only did not kill, but the decreased levelsof cytotoxicity indicate that they blocked the ability of other T cellsto kill blood cells of the recipient.

Example 3 Treating a Patient with Chronic Myelocytic Leukemia with theStem Cells from a Histoincompatible Donor: Tolerization with Mitogens

The harvested PBMC of the donor are placed in a sterile container inHBBS as in Example 1. The cells are then incubated with mitogens toinduce lymphocytes to become non-responsive to histocompatibilityantigens of the recipient. In this case the cells are incubated withphysiological concentrations of concanavalin A (Con A) for 4 to 72 hoursusing standard incubation techniques. The concentration of Con A usedcan range from about 0.01 to about 10 micrograms/ml with 1 microgram/mlbeing presently preferred. Alternatively, SEB may be used as the mitogenat concentrations of 0.001 to 100 ng/ml.

The incubation of the mononuclear cells in the mitogen solutionincreases the population of T suppressor cells. These cells, whentransferred to the recipient, will enable the stem cells to engraftwithout causing GVHD. Although it is not known how the mitogens work, itis believed to induce the production of TGF-beta by certain mononuclearcells in preparation, and the TGF-beta acts on T cells to becomesuppressor cells.

After the cells have been incubated with the mitogens, the cells arewashed with HBBS to remove any mitogens that are in the solution. Thecells are suspended in 200-500 ml of HBBS, mixed with the stem cells andadministered to a patient with CML who has been treated withmyeloablative agents to prepare the stem cells for engraftment.

Once the donor hematopoietic cells lymphocites engraft in the recipient,and the patient again becomes healthy and free of leukemic cells. If theleukemic cells recur, the patient receives a transfusion of donorlymphocites and the leukemic cells again disappear.

Example 4 Treating a Patient with Chronic Myelocytic Leukemia with theStem Cells from a Histoincompatible Donor: Tolerization with Anti-CD2Monoclonal Antibodies

The harvested enriched stem cell preparation of the donor are placed ina sterile container in HBBS as in Example 1. The cells are thenincubated with anti-CD2 monoclonal antibodies to induce lymphocytes tobecome non-responsive to histocompatibility antigens of the recipient.In this case, the cells are incubated with anti-CD2 monoclonalantibodies for 4 to 72 hours using standard incubation techniques. Theconcentration of anti-CD2 monoclonal antibodies are 10 ng/ml to 10ug/ml. Optionally, 1-1000 units of IL-2 can be added.

The incubation of the mononuclear cells in the anti-CD2 solutionincreases the population of T suppressor cells. These cells, whentransferred to the recipient will enable the stem cells to engraftwithout causing GVHD. It is believed that incubation with anti-CD2monoclonal antibodies induces the production of TGF-beta by certainmonuclear cells in preparation, and the TGF-beta acts on T cells tobecome suppressor cells.

After the cells have been incubated with the anti-CD2 monoclonalantibodies, the cells are washed with HBBS to remove antibodies that arein the solution. The cells are suspended in 200-500 ml of HBBS mixedwith the stem cells harvested previously and administered to a patientwith CML who has been treated with myeloblative agents to prepare thestem cells for engraftment.

Once the donor hematopoietic cells lymphocytes engraft in the recipient,and the patient again becomes healthy and free of leukemic cells. If theleukemic cells recur, the patient receives a transfusion of donorlymphocytes and the leukemic cells again disappear.

Example 5 Treating a Patient with Chronic Myelocytic Leukemia with theStem Cells from a Histoincompatible Donor: Tolerization with Mitogensand Cytokines

The harvested PBMC of the donor are placed in a sterile container HBBSas in Example 1. The cells are then incubated with cytokines andmitogens to induce lymphocytes to become non-responsive tohistocompatibility antigens of the recipient. In this case the cells areincubated with physiological concentrations of Con A or SEB, IL-2 orIL-10 and TGF-beta for 4 to 72 hours using standard incubationtechniques.

After the cells have been incubated with the cytokines and mitogens, thecells are washed with HBBS to remove any cytokine and mitogen that arein the solution. The cells are suspended in 200-500 ml of HBBS mixedwith the stem cells and administered to a patient with CML who has beentreated with myeloabative agents to prepare the stem cells forengraftment.

Once the donor hematopoietic cells and lymphocytes engraft in recipientand the patient again becomes healthy and free of leukemic cells. If theleukemic cells recur, the patient receives a transfusion of donorlymphocytes and the leukemic cells again disappear.

Example 6 Treating a Patient with Chronic Myelocytic Leukemia with theStem Cells from a Histoincompatible Donor; Tolerization with a Mitogenand Cytokine

The harvested PBMC of the donor are placed in a sterile container inHBBS as in Example 1. The cells are then incubated with a cytokine and amitogen to induce lymphocytes to become non-responsive tohistocompatibility antigens of the recipient. In this case the cells areincubated with physiological concentrations of ConA, and IL-2 for 4 to72 hours using standard incubation techniques. In another case, SEBcould be used.

After the cells have been incubated with the cytokines and mitogens, thecells are washed with HBBS to remove any cytokine and mitogen that arein the solution. The cells are suspended in 200-500 ml of HBBS mixedwith stem cells and administered to a patient with CML who has beentreated with myeloablative agents to prepare the stem cells forengraftment.

Example 7 Treating a Patient with Chronic Myelocytic Leukemia who hasDeveloped GVHD Following the Stem Cell Transplant

In the instance that the initial procedure to prevent early or late GVHDfollowing the stem cell transplant is not successful, this event will bemanaged BY transfer of a larger number of donor T cells that have beenconditioned to become suppressor cells. Approximately 1×10⁹ PBMCsobtained by leukopheresis are concentrated in a sterile leukopak; inHanks balanced salt solution (HBBS). The PMMCs or separated CD8+ T cells(or the specific suppressor cell precursor subset CD8+CD45RA+C27+)prepared by immunoaffinity columns will be treated with antiCD2monoclonal antibodies and/or mitogens and/or cytokines described aboveto condition them to become suppressor cells.

After incubation with the cytokines or mitogens for a period of timeranging from 4 hours to 72 hours, the cells are washed to remove thecytokines or mitogens and then are transferred to the recipient. Theseconditioned T cells migrate to lymphoid organs and suppress the GVHD.

Besides chronic myelocytic leukemia, other hematologic malignancies suchas acute and chronic leukemias, lymphomas, solid tumors such as breastcarcinoma or renal cell carcinoma among a few, and non-malignantdiseases such as severe anemias (thalassemia, sickle cell anemia) can betreated with mismatched allogeneic stem cells.

Another aspect of this invention is a kit to perform the cell incubationwith the cytokines. The kit comprises a sterile incubating containerwith the appropriate concentration of cytokines preloaded within thecontainer. In one embodiment of the kit, the cytokines are present inlyophilized form in the container. The container is preferably a bag,similar to an IV bag. The lyophilized cytokines are reconstituted withHBBS and then the cells are injected into the container and thoroughlymixed and incubated. In another embodiment of the invention thecytokines are already in solution within the container and all that hasto be done is the injection of washed stem cell preparation andincubation.

What is claimed is:
 1. A method for treating donor cells to ameliorategraft versus host disease in a recipient patient comprising: a) removingperipheral blood mononuclear cells (PBMC) from a donor; b) treating saidcells with a suppressive composition comprising TGF-β for a timesufficient to induce T cell tolerance; and c) administering said cellsto said patient.
 2. A method according to claim 1 wherein saidsuppressive composition firther comprises IL-10.
 3. A method accordingto claim 1 wherein said method further comprises adding said cells todonor stem cells prior to administering to said patient.
 4. A methodaccording to claim 1 wherein said suppressive composition firthercomprises IL-2.